**4.1 Laboratory techniques and protocols**

Different instrumental analytical techniques were reported by scientists as tools to identify and quantify antioxidants in water and hydroalcoholic extracts obtained from different grapes anatomic parts, and also for genetic characterization [3, 4, 19, 36, 71, 73, 75–78]. Top instrumental techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), with various detection devices are used to obtain detailed information on the bioactive compounds profile and content, or on genetic information (geographical mapping etc). Spectroscopic techniques like ultraviolet–visible (UV–VIS), Fourier transform infrared (FTIR) and Raman, are widely used to establish the antioxidant activity of grapes samples, to identify and/or quantify classes of antioxidant species (*i.e.* polyphenols, flavonoids, etc.) and other bioactive compounds, as well as to provide raw entry data for chemometric analysis. Also, rapid electrochemical tests (*i.e.* pH, conductivity) or refraction index measurements are used to evaluate either the acidity, total dissolved solids, or total dissolved sugars in grape based samples.

Antimicrobial activity is an important characteristic for any material intended to be used in applications related to health, food or others [19, 72]. In this study, disc diffusion assay and minimum inhibitory concentration methods have been used to evaluate this property of red grape extracts against some bacterial strains isolated from natural environment, some important conclusions have been drawn and were presented below.

Considering the large-scale application of developed laboratory protocols, grapes samples were mainly characterized through spectroscopic methods such as absorption techniques of UV–VIS and FTIR, and Raman scattering. These techniques are routinely used in laboratories, and generally accepted as providing cost-effective, rapid measurements, with a convenient sample treatment, or non-destructive. Even if the recorded spectra are often not readily useable, and need data processing and analysis, further use of chemometrics may help to extract meaningful conclusions from multivariate data.

Analytical protocols included the classic steps of sampling, sample preparation, and qualitative and/or quantitative analysis. For the sampling step, grapes samples of four varieties were harvested from Romanian vineyards (out of which one was a native wine variety) as described in previous published works [3, 4, 19, 75], and then representative portions from each sample were taken for further treatment. The four varieties studied were Merlot, Pinot Noir, Feteasca Neagra and Muscat Hamburg. Grape skins and seeds were dried in the oven at 40°C for 48 hours and then stored at room temperature in closed vials, while the pulp fraction was frozen and maintained at - 18°C, and defrosted in the day of laboratory tests. To obtain the grape extracts, classic maceration and ultrasound assisted extraction procedures have been applied, both at room temperature, and using either deionized water (<0.05 μS/cm) or hydroalcoholic (50%, v/v) solvents, for a total extraction time of 24 hours. For maceration, magnetic stirring at 150 rpm has been applied for the first 3 hours, and for the second method, the ultrasound field of 45 kHz has been applied for the first 30 minutes. Then, for the remaining time up to 24 hours, samples rested at room temperature, in dark and non-humid atmosphere. For dry grape skins and seeds samples a 4% (dry weight/volume dw/v) ratio was used, while for the pulp samples, the grape fraction to solvent volume was of 12% (w/v). In general, extractions using 50 mL of solvent were proved sufficient for one set of

analysis. Separation of liquid and solid fractions was performed by centrifugation at 1000 rpm, for 10 minutes, and filtration (Whatman 4).

#### *4.1.1 UV–VIS spectroscopy*

This technique uses the interaction of the light with wavelengths in the range 200–800 nm with the molecules existing in the material of interest. An absorption phenomenon appears, with non-bonding and π-bonding electrons provide the strongest absorbances. Aromatic molecules, antioxidants such as phenolic molecules, flavonoids in particular are examples of molecules where UV–VIS spectroscopy may be successfully applied. The method is considered to have a limitation in sensitivity, because of the inability to differentiate between molecules absorbing in the same wavelengths range. Samples are either scanned as they are, or prepared according to specific protocols indicating qualitative or quantitative determinations.

Antioxidant activity (AA), total polyphenols content (TPC) and total flavonoids content (TFC) have been determined in this study, by using UV–VIS spectroscopy.

**Table 1** shows examples of antioxidant compounds that may be present in grapebased samples [4, 36, 75, 77]. As may be observed, the general structure of polyphenols contains at least one aromatic ring, with at least one hydroxyl group bonded on it. These compounds are classified considering the number of rings and the functional groups bound in the structure, and thus there are: phenolic acids, flavonoids, stilbenes, and lignans, coumarins, tannins. The health benefits of bioactive phenolic compounds have been demonstrated, and their contribution to the wine quality in terms of sensory perception (color, taste, mouthfeel, flavor, astringency, bitterness) have been recently discussed in detail [79].

With respect to flavonoids structure, in **Figure 2** one may observe that it contains two benzene rings (A and B) and an oxygen containing pyran ring (C). Flavonoids' classification in six subclasses is generally accepted, and the difference between them is given by the oxidation level of the C ring of the basic 4-oxoflavonoid (2-phenyl-benzo-γ-pyrone) nucleus, and thus there are: flavanols, flavones, isoflavones, flavanones, anthocyanidins and flavonols. **Table 1** shows the example of quercetin which belong to flavonols sub-class. The antioxidant activity of flavonoids, as for polyphenolics in general, is due to the presence and position of the multiple hydroxyl groups in their structure.

In the following paragraphs, the analytical protocols applied to generate quantitative phytochemical data of studied grape samples will be provided.

*Total polyphenols content (TPC)* was determined through Folin Ciocalteu method [80], the procedure was slightly adapted for grapes samples as prepared in the present study [3, 4, 19, 75]. Folin Ciocalteu reagent consists of a mixture prepared by dissolving sodium tungstate (Na2WO4**·**2H2O) and sodium molybdate (Na2MoO4**·**2H2O) in water, and adding hydrochloric acid and phosphoric acid. Commercial already prepared reagent may be also procured. The chemical process, occurring at basic pH, is based on molybdenum reduction from +6 (yellow) to +4 (blue) after the oxidation of polyphenols in samples. The light absorption of a monochromatic radiation of 765 nm was measured with an UV–VIS spectrophotometer. Colored liquid samples were placed in glass cuvettes with 10 mm light-path, readings were done vs. a blank sample prepared with all reagents as samples, but with extraction solvent instead of grapes extract. The calibration curve has been plotted before each measurement set of samples, with gallic acid as reference antioxidant in the concentration range of 0.01–0.08 mg/mL. Similar experimental procedures were applied for both aqueous and hydro-alcoholic extracts, different samples dilutions were used so that the linear domain of Beer–Lambert–Bouguer

*Romanian Organic and Conventional Red Grapes Vineyards as Potential Sources… DOI: http://dx.doi.org/10.5772/intechopen.98972*

**Figure 2.** *General structure of flavonoids and their subclasses.*

law and calibration range were reached. Final results were provided as total polyphenols content (TPC) expressed as milligrams of gallic acid equivalents per mL of grapes extract, and then reported to dry weight (mg GAE/g d.w.). All experiments were performed in triplicates and the means ± standard deviations (SD) were reported [3, 4, 19, 40, 75].

*Total flavonoid content (TFC)* in grapes fractions extracts was determined through the aluminum chloride colorimetric assay described in previous papers [19, 75]. In this method, some complex combinations form as products of the reaction between the aluminum ions and the carbonyl group from C-4 carbon, and hydroxyl groups from C-3 or C-5 carbons from flavonoids structure. In addition, other chemical bonding may appear between the aluminum ions and the ortho-dihydroxyl groups from A- and B- nucleus of flavonoids. All these chemical processes lead to a yellow color of the working solution, and thus the spectrometric measurement was performed at a wavelength of 510 nm, in glass cuvettes. Deionized water was used for the instrument baseline, and a calibration curve has been plotted in the range of 0.1–1 mg/mL using quercetin as reference flavonoid. Total flavonoids contents were provided as mg quercetin equivalents per mL grape fraction (skin, etc) extract. Calculations to convert the total flavonoids content in the solid grapes samples may be performed for each studied grape fraction, when needed. Analytical data were collected on triplicate samples, mean values together with standard deviations were reported [3, 4, 19, 40, 75].

*Antioxidant activity (AA)* of grapes extracts was evaluated by using the method involving formation of a phosphomolybdenum complex compound, and optical densities were measured at 700 nm, in glass cuvettes with 10 mm optical path [81]. The choice of Prieto procedure was a consequence of some unsatisfactory results obtained for skin extracts when applying the 2,2-diphenyl-1-picrylhydrazyl DPPH• assay, one of the most frequently used method. It was considered that color interferences are the reason this unsuitability; as known, the DPPH• assay involves monitoring the decrease in color intensity of a purple reagent, while the tested samples (*i.e.* red grapes skin extracts) had colors in the same spectral range.

#### *4.1.2 Vibrational spectroscopy*

Two vibrational spectroscopic techniques were used during experiments, the infrared (IR) light absorption and Raman scattering, both aiming at investigating the chemical functional groups of organic compounds in studied grape samples, and potential changes occurring while applying extraction procedures. Gathering information on differences between grapes sampled from organic and conventional vineyards was also in the scope of this study.

The Fourier Transform infrared (FTIR) spectrometer used was Vertex 80v (Bruker) equipped with diamond attenuated total reflection (ATR) crystal accessory, and samples were placed on the measurement position without any additional preparation. The absorption frequencies were recorded in the mid infrared range of 4000–400 cm−1, the average spectrum of 32 scans (with baseline and atmospheric correction), was declared an experimental result, and considered for further data processing. Same IR scanning procedure was followed for each of the studied samples.

Raman spectra for studied samples have been recorded with a Xantus 2 (Rigaku) spectrometer, using a light source of 1064 nm, at a power of 490 mW. The average of 5 scans (with baseline correction) was taken as the experimental result for each sample, and presented as intensity vs. Raman shift in the wavenumber range of 2000–200 cm−1.

#### *4.1.3 Antimicrobial activity determination*

To evaluate antimicrobial activity of the grapes extracts, observation and quantification of the growth of several strains of bacteria isolated from natural environments during their contact with studied samples. Both disc diffusion and minimum inhibitory concentration assays were applied [3]. First, several bacterial strains were isolated from different habitats, grown in agar meat broth, and incubated at 37 ± 0.2°C, then characterized by classical microbiological techniques. These bacterial cultures were used to prepare inocula for the antimicrobial testing, colonies from 24 h-old plates were picked, suspended in appropriate media, and aerobically grown at 37°C for 24 h. It worth mentioning at this point that all the operations related to antimicrobial activity determination were performed according to a lab-protocol that avoided contamination (*i.e.* manipulations under UV light, etc).

For the disc diffusion method, a volume of 20–50 μL of fresh bacterial culture with the optical density at 600 nm between 0.2 and 0.4 was spread on Petri dishes with the media. Sterile 6 mm paper disks were impregnated in the grape extracts for 1 h, then placed on the Petri dish at approx. 15 mm from edge, and at 30 mm distance between each other, and in the end incubated at 37 ± 0.2°C for 2 days. One considers a sample as having antimicrobial activity, if after the above-mentioned incubation time, a clear area (halo) may be observed on the inoculated Petri dish around the disk impregnated with the respective sample.

The minimum inhibitory concentration of grape extracts was determined as the lowest concentration of the sample that completely inhibited the growth of tested microorganisms, as visually detected by the normal human eye. The incubation time considered was 48 h at 37 ± 0.2°C, and control samples without grape extract were tested in each set of experiments.

## *4.1.4 Chemometric methods*

In is well known that chemometrics is generally applied to provide additional information to the direct interpretation of experimental data collected through various laboratory techniques. The usefulness of chemometrics may arise from both its descriptive approach *(i.e.* finding relationships and structure of the systems), and from the predictive one (modeling of some chemical properties, so that new properties or specific behavior may be predicted).

Several chemometric methods have been applied during the study, as valuable tools aiming at a further interpretation of the instrumental analytical data. In this respect, we may list herein the multiple linear regression, bivariate correlations of data (on the basis of Pearson coefficients), and the SPSS classification through hierarchical cluster analysis. Also, multivariate analysis and corresponding methodologies have been applied to process large data sets generated by the vibrational spectroscopic used for samples characterization [82]. Other techniques like principal component analysis (PCA), agglomerative hierarchical clustering (AHC) and discriminant analysis (DA) were also applied in this study [38, 39, 83–85], as well as combinations between them [70, 82, 86].

The Statistical Package for the Social Science v24.0 software for MS Windows (SAGE IBM® SPSS®) was used when measured phytochemical parameters and antimicrobial activity were taken into consideration for data analysis. The significance of differences between various experimental groups was evaluated at 5% level of significance.

For statistical analysis of spectral data, the XLSTAT software, 2021.1.1 version has been used (©Addinsoft, USA). First, Box-Cox transformation [82, 87, 88] was applied to obtain approximately normally distributed values. Then, principal component analysis (PCA) was used to reduce the dimensionality of the spectral data to a smaller number of components. The analysis of the score plots (FTIR and Raman data) for the first three principal components (PCs) was based on the partial bootstrap method [89], in order to estimate the proximity between the observations and to know which observations are significantly different from each other. Agglomerative Hierarchical Clustering (AHC) was performed using the Euclidean distance as the distance measure and single linkage (Ward's method) strategy to link clusters within the data set [76]. Discriminant Analysis (DA) was applied considering that when the number of variables exceeds the number of samples, one method of multivariate discrimination is to use principal components analysis and then to perform canonical variates analysis [83, 84]. Combining both PCA and DA approaches, in so called PC-DA model, leads to improving the efficiency of classification, as this procedure automatically finds the most diagnostically significant features [85, 86, 90].

Beyond the technical details of their specific application on the data recorded by laboratory and instrumental techniques, these chemometric methods aimed to complete the direct interpretation of the analytical results. Thus, additional information regarding potential correlations between the potential valuable compounds that may be extracted from studied grape samples, their antimicrobial activity, and the vineyard management type, grape varieties, or grapes anatomic parts used to prepare the studied extracts, etc. was of a significant interest once one started to apply the chemometrics.
